JPH1025300A - New polypeptide, antibody capable of recognizing the same and use of the antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new polypeptide having a specific amino acid sequence, with its expression induced in its reoxygenation after subjected to low oxygen load, with its DNA and antibody useful for the diagnosis, assessment and morbid state elucidation of, and for developing preventive/therapeutic agents for ischemia/reperfusion disorders. SOLUTION: This new polypeptide has amino acid sequence virtually shown by the formula, with its expression induced in its reoxygenation after subjected to low oxygen load. Examination of its expression enables the diagnosis, assessment and morbid state elucidation in vitro and/or in vivo of ischemia/reperfusion disorders, and use of the antibody against this polypeptide enables the screening of preventive/therapeutic agents for ischemia/reperfusion disorders. This polypeptide is obtained by the following process: primary-cultured rat astrocytes are cultured until they come to a confluent state, and then subjected to low-oxygen load treatment followed by reoxygenation treatment; the mRNA of the resultant astrocytes is separated, and a cDNA library is constructed using the mRNA as template followed by cloning and then expression in host cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel polypeptide whose expression is induced during reoxygenation after hypoxic load, especially during reperfusion after ischemia, a DNA encoding the same, and a recombinant vector containing the DNA. And a host cell transformed with the recombinant vector, and the novel polypeptide-specific antibody and its use.

[0002]

2. Description of the Related Art Death caused by ischemic state of the brain such as stroke or cerebral infarction is still the leading cause of death after death due to cancer. Not only the death of nerve cells during cerebral ischemia, but also delayed neuronal death occurring within one to two weeks after ischemia is an important issue in the treatment of current brain disorders. Also, cessation of blood flow is a serious problem in organs other than the brain. Organ damage caused by ischemia / reperfusion occurs as a result of cellular responses to two environmental changes: hypoxia due to abrupt cessation of blood flow (Hypoxia) and subsequent reoxygenation due to reperfusion. Hypoxia has a sustained major effect on changes in cell behavior in ischemic organ injury (Matsuo et al., J. Biol.
Chem. (1995) 270, 28216-28222). Also, in cultured cells, the loss of oxygen in the atmosphere causes a change in energy metabolism in the cell's metabolic system (Loke et al., Am. J. Physiol.
(1992) 263, C326-C333, Maeda et al., J. Exp. Med. (199
4) 180, 2297-2308), enhanced glucose metabolism (Hori et al.,
J. Neurochem. (1994) 62, 1489-1495), oxygen regu
Expression of stress proteins that are related proteins (ORPs) (Ogawa et al., J. Clin. Invest. (1990) 85, 1090-109
8) cause characteristic changes such as: These changes are
It is considered to be a defense mechanism of cells against environmental changes that threaten cell survival.

[0003] Compared with the cellular response to hypoxic load due to ischemia, the cellular response during reoxygenation that occurs during reperfusion after ischemia in vivo has not been studied so far. From some findings, the cellular response to reoxygenation is
Rather than merely passive self-protection against elevated oxygen levels, it has a more active role in promoting remodeling of tissue after ischemia, and has the potential to return to an oxygen-rich environment during ischemia / reperfusion. Although it is suggested that new protein synthesis is necessary for adaptation, at the time of ischemia / reperfusion, especially for proteins expressed at the early stage of ischemia / reperfusion or genes encoding them, Very few concrete elucidations have been made.

[0004]

An object of the present invention is to provide a novel polypeptide whose expression increases at an early stage of ischemia / reperfusion and a gene encoding the novel polypeptide. Another object of the present invention is to provide a recombinant vector and a transformant using the gene. Another object of the present invention is to provide a method for producing the above novel polypeptide using the transformant. Also,
Another object of the present invention is to provide an antibody exhibiting affinity for the polypeptide and its use.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies to achieve the above object, and as a result, cloned a novel gene whose expression is remarkably increased at an early stage of reoxygenation after hypoxic load, and The nucleotide sequence and the amino acid sequence of the polypeptide encoded by it were determined. The present inventors have further studied and found that the novel polypeptide of the present invention is very likely to be a vesicle transport-related factor. In addition, it was confirmed that the expression was induced in vivo at an early stage of reperfusion, as in the brain tissue of a rat cerebral ischemia / reperfusion model. Furthermore, based on these findings, the present inventors prepared a specific antibody against the polypeptide, evaluated the ischemia / reperfusion injury model using the antibody, and prevented / treated for ischemia / reperfusion injury The present invention has been completed by completing a drug screening method and the like.

That is, the present invention is described below. (1) A novel polysaccharide having substantially the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing, and whose expression is induced upon reoxygenation after hypoxic load such as reperfusion after ischemia. Peptides, especially the novel polypeptides described above, which are derived from mammals, especially rats. (2) DN encoding the novel polypeptide of (1) above
A, in particular, a DNA substantially having at least the nucleotide sequence of at least nucleotides 14 to 1924 in the nucleotide sequence of SEQ ID NO: 2 in the Sequence Listing. (3) A recombinant vector containing the DNA of (2). (4) A host cell, particularly an animal cell, transformed with the recombinant vector of (3). (5) A method for producing the novel polypeptide, comprising culturing the host cell of (4) and collecting the novel polypeptide of (1) from the resulting culture. (6) an antibody exhibiting affinity for the polypeptide of (1),
In particular, all or part of the amino acid sequence substantially as shown in SEQ ID NO: 1, preferably at least amino acid number 3
An antibody exhibiting affinity for a peptide having the amino acid sequence represented by any one of 46 to 364. (7) A method for evaluating an ischemia / reperfusion injury model, comprising using the antibody of (6). (8) A kit for evaluating / diagnosing ischemia / reperfusion injury containing the antibody of (6) above. (9) A method for screening a drug for preventing and treating ischemia / reperfusion injury, which comprises using the antibody of (6) above;
And a kit therefor.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The novel polypeptide of the present invention is transiently expressed during reoxygenation after hypoxic load, particularly at an early stage of reoxygenation, particularly within one hour after the start of reoxygenation. It is characterized by being guided.

The mode of hypoxia and subsequent reoxygenation is not particularly limited, but in vivo, ischemia / reperfusion is a typical example. In the present invention, ischemia / reperfusion refers to a process in which a tissue restores a normal oxygen state by reperfusion after a hypoxic state due to the cessation of blood flow, and whatever causes the cause. Good. For example, cerebral ischemia and subsequent reperfusion due to cerebral infarction, ischemia of various organs and subsequent reperfusion due to thrombus, etc., as well as temporary ischemia during surgery such as organ transplantation and reperfusion after surgery etc. No.

[0009] In the case of cultured cells, the culture is returned to normal pressure after culturing in a hypoxia chamber, so that low oxygen load and
A reoxygenation condition can be created. For example, examples of hypoxia load / reoxygenation in primary cultured astrocytes of rats include the following methods. Place the confluent cultured cells in a hypoxic chamber,
The cells are exposed to hypoxia for 24 to 48 hours so that the dissolved oxygen concentration in the medium is 6 to 10 torr, and then the cells are re-oxygenated by returning them to normal gas again.

The novel polypeptide of the present invention has an amino acid sequence substantially as shown in SEQ ID NO: 1 in the Sequence Listing. The amino acid sequence has about 38% homology to Sly1p, a vesicle transport protein between the endoplasmic reticulum and the Golgi apparatus in yeast. Furthermore, it has many domains homologous to other yeast vesicle transport proteins, Sec1p, Vps45p, and Vps33p.
It also has homology to vesicle transport proteins such as op and rat n-Sec1. This fact and the localization in the Golgi suggest that the novel polypeptide of the present invention may be a vesicle transport-related factor between the endoplasmic reticulum and the Golgi (see Test Example 3 and FIG. 5).

The amino acid sequence is not particularly limited as long as it does not change the physicochemical, biochemical, and biological properties such as the three-dimensional structure, antigenicity, expression mode, and intracellular localization. May have a substitution, deletion, insertion or modification.

The origin of the novel polypeptide of the present invention is not particularly limited, but is preferably a mammal, such as a mouse,
Cells or tissues of rats, rabbits, sheep, cows, horses, monkeys, humans, etc., and more preferably cultured cells or tissues of rats. Primary cultured astrocytes are exemplified as cultured cells, and brain tissues, testis tissues, and the like are exemplified as tissues.

The novel polypeptide of the present invention can be produced by appropriately using a known method such as a method of extracting and purifying animal tissues or cells as a raw material, a method of chemically synthesizing or a gene recombination technique. Preferably, a method of cloning DNA encoding the polypeptide and then culturing a host cell transformed with a recombinant vector containing the cloned DNA to collect the polypeptide is exemplified. As a preferred cloning method, a cDNA library is prepared from cultured cells or tissues before and after ischemia / reperfusion, respectively, by utilizing the property that the expression of the polypeptide is remarkably increased during ischemia / reperfusion. Subtraction method, PC using cDNA as template
Differential display method for comparison by the R method (Matsuo et al., J. Biol. Chem. (1995) 270, 28216-2822)
There is a method using 2).

The DNA of the present invention is a DNA having a nucleotide sequence encoding the amino acid sequence of the novel polypeptide of the present invention.
Although it is not particularly limited as long as it is A, preferably, DNA encoding the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, more preferably at least base numbers 14 to 1924 in the base sequence substantially shown in SEQ ID NO: 2 in the sequence listing Is a DNA having a base sequence represented by Where "substantially"
Is a DNA containing an intervening sequence (intron) that can be spliced to form mRNA corresponding to the nucleotide sequence represented by nucleotide numbers 14 to 1924 when transcribed in an appropriate host cell, for example, a mammalian cell. Is also meant to be included.

[0015] The DNA of the present invention may be obtained by any method. For example, complementary DNA (cDNA) prepared from mRNA, genomic DNA prepared from a genomic library, DN synthesized chemically
A, DNAs obtained by amplifying by PCR method using RNA or DNA as a template, and DNAs constructed by appropriately combining these methods are also included.

For example, by utilizing the property that the expression of the polypeptide of the present invention is remarkably increased during ischemia / reperfusion, a differential display method in which cDNAs are prepared from cultured cells or tissues before and after reperfusion and compared, respectively. The following method is exemplified as the cloning method used.

First, for example, guanidine thiocyanate method, hot phenol method, acid guanidine-phenol-chloroform (AGPC) can be obtained from each of the primary cultured astrocytes derived from hypoxia-loaded and post-hypoxia-reoxygenated rats. ) Using a known method such as
Extract A. The obtained total RNA is RT-PC
Alternatively, the poly (A) ⁺ RNA may be purified by affinity chromatography using oligo (dT) cellulose, poly-U-sepharose, or the like.

Next, a cDNA is synthesized by a known method using a reverse transcriptase using the obtained RNA as a template, and then amplified by PCR using the obtained RNA as a template and both upper and lower primers as primers. Oligonucleotides used as primers are not particularly limited, but 12 to 25 m
It is desirable to use one having a length of about er. The amplified products are separated by electrophoresis and stained with ethidium bromide. The gel portion containing the cDNA band that has been specifically amplified in the reoxygenated astrocytes is cut out and extracted. The extracted cDNA fragment is further reamplified by performing an amplification reaction under the same primers and under the same reaction conditions. The reamplified cDNA fragment is treated with an appropriate restriction enzyme, if necessary, or a linker DNA is added and inserted into an appropriate cloning vector. Although the cloning vector is not particularly limited, for example, pBR33
2, pUC119, pBluescript and the like.

The cDNA thus cloned
The nucleotide sequence of the fragment can be determined by the known Maxam-Gilbert method, dideoxy method, or the like. Further, using the cDNA fragment as a probe, for example, rat cDN
CD of full length polypeptide of the invention from A library
NA can be obtained.

The recombinant vector of the present invention is not particularly limited as long as it can replicate and maintain or autonomously propagate in various prokaryotic and / or eukaryotic host cells, and includes plasmid vectors and phage vectors. The recombinant vector can be conveniently prepared by operably linking a DNA encoding the polypeptide of the present invention to an expression vector available in the art by a conventional method. The expression vector to be used is not particularly limited as long as it has a function of expressing and producing the polypeptide of the present invention in various prokaryotic and / or eukaryotic host cells. It preferably contains a selectable marker gene. For example, when transforming mammalian cells, animal viruses such as S
V40, RSV, MMLV and other promoters and a polyadenylation signal are ligated to a plasmid linked via a restriction enzyme site, preferably a multiple cloning site.
Plasmids into which a selection marker gene (neomycin resistance gene, dihydrofolate reductase, etc.) derived from a plasmid such as V2-neo or pSV2-dhfr can be used.

The transformed cell of the present invention can be prepared by introducing an expression vector containing a DNA encoding the above-described polypeptide into a host cell. The host cell is not particularly limited as long as it is compatible with the expression vector to be used and can be transformed, and various cells such as natural cells or artificially established recombinant cells usually used in the technical field of the present invention can be used. Although available, cells that do not express the endogenous polypeptide or that lack the gene encoding the polypeptide are more preferred. Specific examples include bacteria such as Escherichia coli and Bacillus subtilis, fungi such as yeast, animal cells and insect cells, and the like. Preferably, mammalian cells, especially rat-derived cells, hamster-derived cells (C
HO, BHK, etc.), mouse-derived cells (COP, L, C1)
27, Sp2 / 0, NS-1, NIH T3, etc.), monkey-derived cells (COS1, COS3, COS7, CV1, V
elo) and human-derived cells (Hela, diploid fibroblast-derived cells, myeloma cells, Namalwa, etc.)
Is mentioned.

The introduction of the expression vector into the host cell can be performed by a conventionally known method. For example, when introduced into mammalian cells, a calcium phosphate coprecipitation method, a protoplast fusion method, a microinjection method, an electroporation method, a lysosomal method and the like can be mentioned.

The novel polypeptide of the present invention can be produced by culturing a transformed cell containing the expression vector prepared as described above. The medium preferably contains a carbon source, an inorganic or organic nitrogen source necessary for the growth of the host cell (transformant). Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose. Examples of the nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extracts, soybean meal, potato extract, and the like. Is exemplified. If desired, other nutrients (eg, inorganic salts (calcium chloride, sodium dihydrogen phosphate, magnesium chloride, etc.), vitamins, antibiotics (tetracycline, neomycin, kanamycin, ampicillin, etc.)) may be included.

The culturing is performed by a method known in the art. Culture conditions such as temperature, pH of the medium, and culture time are appropriately selected so that the polypeptide is produced in large quantities.

For example, when the host is an animal cell, the medium may be, for example, about 5 to 20% fetal calf serum (FCS).
Essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), RPMI-1640 medium, 199
A medium or the like can be used. The pH of the medium is preferably about 6 to 8, and the culture is usually carried out at 30 to 40 ° C. for about 15
This is performed for up to 72 hours, and if necessary, aeration and stirring can be performed.

The novel polypeptide of the present invention can be obtained from the culture obtained by the above culture, for example, by the following method. First, the culture is subjected to a conventional method such as filtration or centrifugation to collect the cells, suspended in a suitable buffer, and further added with a surfactant at a suitable concentration to solubilize the membrane. When the host cell has a cell wall, pretreatment with lysozyme or ultrasonic waves is required. Examples of the surfactant include sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB) and the like. Since these have a strong protein denaturing action,
In order for the protein to fold so as to have biological activity, it is preferable to use a mild nonionic surfactant such as, for example, Triton X-100. Next, the polypeptide is isolated and purified by appropriately combining generally used methods with the obtained crude extract in the presence of a surfactant, if necessary. For example, methods using solubility such as salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, methods using differences in molecular weight such as SDS-PAGE,
Methods using charge such as ion exchange chromatography, methods using specific affinity such as affinity chromatography, methods using difference in hydrophobicity such as reverse phase high performance liquid chromatography, isoelectric focusing, etc. A method using the difference between isoelectric points is exemplified.

The antigen used for preparing an antibody having an affinity for the novel polypeptide of the present invention is not particularly limited as long as its antigenicity is not changed. Specifically, the novel polypeptide can be used as the antigen. Furthermore, a peptide having substantially all or a part of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, preferably at least amino acid 3 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing
A peptide having an amino acid sequence represented by any one of 46 to 364 is used. The polypeptide and / or peptide can be obtained by appropriately using a known method such as a method of extracting and purifying from animal tissue as a raw material, a method of chemically synthesizing, and a gene recombination technique.

Specifically, the method of chemically synthesizing is a method using a peptide synthesizer, and the method of recombining is to incorporate DNA encoding the polypeptide into an expression vector, and to use the recombinant vector for prokaryotic cells. And / or introduction into various eukaryotic host cells, and culturing the resulting transformant.

The immunizing antigen is preferably prepared by mixing with an adjuvant. When the immunity of the antigen is low, it is preferable to use one obtained by binding a carrier protein such as bovine serum albumin (BSA) to the antigen. As an animal used as an object of immunization, for example, mammals such as horses, sheep, goats, rabbits, guinea pigs, and mice, preferably rabbits are used. Immunization is carried out by subcutaneous, intramuscular, intravenous, footpad or intraperitoneal injection of these mammals one or several times. Normally, immunization is performed 1 to 4 times about every 1 to 2 weeks after the initial immunization, and final immunization is further performed about 1 to 4 weeks later. Obtain as serum. A monoclonal antibody can also be prepared by immunizing a mouse, guinea pig, or the like using a conventionally known method. It is preferable that the antibody is appropriately purified and used as necessary. Specific examples of the purification method include ammonium sulfate precipitation, a method using protein A, and a method using an affinity column to which an antigen polypeptide and / or peptide is bound.

The use of the novel polypeptide-specific antibody of the present invention enables a method for evaluating ischemia / reperfusion injury. That is, specifically, it becomes possible to diagnose a pathological condition of ischemia / reperfusion injury, evaluate an ischemia / reperfusion injury model, and the like.

For example, in patients with diseases such as acute renal failure or post-operative organ transplantation, cells obtained by biopsy of organs such as lung, liver and kidney which are predicted to have ischemia / reperfusion injury Used for immunological measurement of tissue. Specifically, after fixing the collected cells with a formaldehyde solution or the like, the specific antibody is used as a primary antibody and reacted with a reagent such as a secondary antibody according to a conventional method to examine the expression status of the novel polypeptide. . Further, a tissue section is prepared using the collected tissue, and the expression state can be examined by the above-described method. In particular, the antibody can be suitably used for investigating and diagnosing tissue and cell damage caused by cessation of blood flow during transplantation of organs such as liver, kidney, and lung, and reperfusion after transplantation.

The effects on various organisms due to ischemia / reperfusion are serious and have been studied by many researchers.
However, to date, at the animal level, large amounts of cells have been simultaneously reduced at the cell level due to factors such as differences in the size of the target animals, differences in health status, and the lack of reproducible ischemia loading means. It is difficult to make a model of ischemia / reperfusion injury based on certain criteria due to factors such as difficulty in exposure to oxygen, and it is extremely difficult to judge whether the model is sufficient, especially It has been. The use of the specific antibody of the present invention enables evaluation of an ischemia / reperfusion injury model. After preparing the model, the antibody is used to confirm the expression status of the novel polypeptide of the present invention, and then used in experiments to obtain a more uniform result in the ischemia / reperfusion injury model. .

Further, by using the specific antibody of the present invention, it becomes possible to screen for a novel preventive and therapeutic agent for ischemia / reperfusion injury. Specific screening methods include the following methods. For example, a hypoxic load / reoxygenation model is prepared using primary cultured astrocytes, and a drug to be tested is applied to the model by appropriately changing the treatment time. Formaldehyde,
After immobilization with an acetone-methanol mixture or the like, the specific antibody of the present invention is used as a primary antibody, and the expression status of the polypeptide is determined by immunofluorescence staining in which the antibody is reacted with a secondary antibody fluorescently labeled with FITC or rhodamine according to a conventional method. Find out. In addition, the drug-treated cells can be solubilized with a surfactant or the like, and the expression status of the polypeptide can be examined by immunoblot analysis using the cell extract. That is, the cell extract is subjected to SDS-PAGE, proteins in the extract are separated, electrically transferred to a nitrocellulose membrane or the like, a specific antibody of the present invention as a primary antibody, and subsequently a secondary antibody and The state of expression of the polypeptide is examined by reaction with a coloring reagent. The effect of the drug can be measured by the presence or absence or the level of expression of the novel polypeptide of the present invention, which is an indicator of ischemia / reperfusion injury.

A kit for evaluating and diagnosing the specific antibody of the present invention,
A known secondary antibody (for example, in the case of immunofluorescent staining, a fluorescently-labeled antibody) may be appropriately used depending on the origin and characteristics of the antibody and the method of measurement when included in a kit for prophylactic and therapeutic drug screening. In the case of immunoblot analysis, an enzyme-labeled reagent is used), and a reagent such as a coloring reagent necessary for the reaction can be included as a set.

[0035]

EXAMPLES The present invention will be specifically described below with reference to examples and test examples, but the present invention is not limited to these examples.

Example 1 cDNA cloning of the novel polypeptide of the present invention from rat primary cultured astrocytes 1) Culture of cells and hypoxia loading / reoxygenation CO ₂ incubator (MCO-175, manufactured by Sanyo)
Matsuo et al. (Matsuo et al., J. Biol. Chem. (1995) 270, 28216-2822) under medium, 37 ° C., 5% CO ₂ conditions.
Rat primary cultured astrocytes prepared according to 2) were placed in MEM supplemented with 10% FCS at 5 × 10 ⁴ cells / cm.
Winding respectively ² cell density 175cm ² culture plates (Falcon) in two, and cultured to confluence. When the cells became confluent, hypoxic load and reoxygenation were performed. The cells were placed in a hypoxia chamber (manufactured by Koi Laboratories), and the concentration of dissolved oxygen in the medium was adjusted to 8 to 10 torr to perform hypoxia load. After 24 hours of exposure to hypoxia, reoxygenation was performed by returning the cells to normal gas. 2
One of the confluent astrocytes is treated with hypoxia (24 hours), and the other is hypoxia / reoxygenated (exposed to hypoxia for 24 hours and then reoxygenated for 1 hour). Processed. The dissolved oxygen concentration in the medium is bl
The results were confirmed with an oad gasanalyzer (manufactured by Radiometer).

2) Preparation of total RNA and cDNA 5 × 1 each of the two types of astrocytes obtained in 1) above
0 ⁸ from TotalRNA Separation
Total RNA was extracted by the AGPC method using Kit (Clontech). Next, cDNA was obtained by a reverse transcription reaction using the RNA sample as a template. All reagents used for extracting cells loaded with hypoxia were equilibrated in a hypoxia chamber. The reverse transcription reaction was performed by the following method. That is, the total RNA obtained above was replaced with DNaseI.
(RNase-free, Boehringer Mannheim) at 37 ° C. for 30 minutes, followed by phenol / chloroform treatment, and 3 μg of each of the treated RNA was added to each of 20 μL.
M dNTP mixture, 2.5 μM oligo dT primer (dT ₁₁ GT) and 300 units of MMLV-derived reverse transcriptase (manufactured by Gibco BRL), an attached reaction buffer and DTT at 37 ° C., 60 ° C. After incubating for 1 minute, single-stranded cDNA was synthesized. In addition, 9
The mixture was heated at 5 ° C. for 5 minutes to dissociate the RNA and cDNA and to inactivate the reverse transcriptase.

3) Differential display method The cDNA obtained above was re-amplified by the method of Liang et al.
ang et al., Science (1992) 257, 967-971). That is, the cDNA obtained in the above 2) (each about 25 n
g) as a template and 1 μM arbitrary prim
er, 200 μM of each dNTP mixture and 2 units of t
Reaction solution 30 each containing aq polymerase (Takara Shuzo)
PCR amplified in μl. The following oligonucleotides were used as both upper and lower primary primers. 5′-TGGTACGGTATA-3 ′ (SEQ ID NO: 3 in the Sequence Listing) The PCR amplification reaction was performed using the Program Temp Cont.
rol SystemPC-700 (made by Astec)
Using 39, 39 cycles of 95 ° C. for 30 seconds (denaturation), 40 ° C. for 1 minute (annealing), 72 ° C. for 1 minute (extension), and 72 ° C. for 5 minutes in the final cycle. After completion of the reaction, the product was electrophoresed on a 14 cm 5% polyacrylamide gel at 100 V for 5 hours and stained with 0.01% ethidium bromide. After observing the gel and reoxygenating, a particularly amplified cDNA band was cut out and boiled with 100 μl of distilled water for 15 minutes. By performing an amplification reaction on the obtained supernatant under the same primers and under the same reaction conditions,
The excised cDNA band was reamplified.

4) Cloning of cDNA Fragment and Determination of Nucleotide Sequence The reamplified cDNA fragment and pT7Blue T-vector (Novagen) were subjected to DNA ligation.
Kit Ver. 2 (manufactured by Takara Shuzo), and then Competent High JM109
The vector incorporating the cDNA fragment was introduced into Escherichia coli JM109 using (Toyobo). Taq D
ye Primer Cycle Sequencen
g kit (Applied Biosystems)
The DNA fragments were sequenced. The sequences of the cDNA fragments were compared for homology in the EMBL and GenBank DNA databases and the NBRF protein database. Furthermore, rat cDNA was prepared using the above cDNA fragment.
Total length 2.1 kb from NA library by the following method
CDNA was obtained. Library is Sprague Dawley ra
t, male, 6 weeks, rat brain cDNA Library, oligo dT
/ Lambda ZAP II (Stratagene)
× 10 ⁶ plaques were plated. The plaque was transferred to a Hybond-N membrane (Amersham), and the membrane was transferred to a hybridization buffer (6 × SSC).
(0.9 M sodium chloride, 90 mM sodium citrate (pH 7.0), 5 × Denhardt mixture, 0.5% SD
S, 100 μg / ml heat-denatured salmon sperm cDNA)
After reacting at 5 ° C. for 3 hours, Random Prime
r Labeling Kit Ver. 2 (manufactured by Takara Shuzo) and hybridized with the cDNA fragment labeled with ³² P-dCTP. The obtained positive plaque was further plated, and the same operation was repeated once again to obtain a single positive plaque. A 2.1 kb cDNA was obtained from the plaque according to the manual attached to the kit, and inserted into pBlueScript II (manufactured by Stratagene). For the 5 'end, 5'-RACE (Rapid Amplification of cDNA End)
The longest sequence was obtained by the method. The obtained cDNA base sequence is shown in SEQ ID NO: 2 in the Sequence Listing, and the amino acid sequence of the polypeptide encoded by the cDNA is shown in SEQ ID NO: 1 in the Sequence Listing. The homology analysis of the amino acid sequence encoded by the obtained cDNA was performed using the BLAST program of GenBank. As a result of homology search, the amino acid sequence had about 38% homology with Sly1p, a vesicle transport protein between the endoplasmic reticulum and the Golgi apparatus of yeast. Se, another vesicle transport protein of other yeasts
It had many domains homologous to c1p, Vps45p, and Vps33p, and also had homology to other vesicle transport proteins Rop (Drosophila), Unc18 (nematode) and n-Sec1 (rat). Therefore, the novel polypeptide of the present invention was considered as one of new vesicle transport proteins.

Test Example 1 Induction of mRNA of New Polypeptide of the Present Invention by Hypoxia / Reoxygenation The induction of the novel polypeptide of the present invention by reoxygenation was confirmed at the mRNA level by Northern blot analysis.
Astrocytes prepared in the same manner as in Example 1 were cultured until they reached a confluent state, and then treated with a low oxygen load (2
4 hours) and astrocytes subjected to low oxygen load / reoxygenation treatment (24 hours of low oxygen load treatment, 15 minutes and 60 minutes after reoxygenation treatment) were prepared. Furthermore, in order to examine the involvement of protein synthesis in the induction of expression by reoxygenation, 10 μg / ml of cycloheximide (a protein synthesis inhibitor) was added 15 minutes before reoxygenation and then reoxygenated for 30 minutes. Active oxygen is usually generated within 5 minutes after reoxygenation. To examine the relationship between this active oxygen and the induction of expression of the novel polypeptide mRNA, 50 μM diphenyliodonium was added 15 minutes before reoxygenation. (DPI, NADP producing active oxygen
H oxidase inhibitor) and reoxygenation to add 3
Astrocytes after 0 minutes were prepared. Total RNA extracted from each astrocyte by AGPC method
g on a 1% agarose / formamide gel.
It was transferred to a mmobilon N membrane (Millipore) overnight. The RNA was immobilized on the membrane by ultraviolet irradiation, and the membrane was reacted at 65 ° C. for 3 hours in the same hybridization buffer used in 4) of Example 1 above. The cDNA fragment of the novel polypeptide of the present invention is
imer Labeling Kit Ver. 2 (manufactured by Takara Shuzo) and labeled with ³² P-dCTP, and reacted in a hybridization buffer at 65 ° C. overnight. Thereafter, the membrane was washed with 2 × SSC / 0.5% SDS and 0.1 × SSC.
The plate was washed with SSC / 0.5% SDS at 65 ° C. and exposed to an X-ray film. The results are shown in FIG. By reoxygenation, mRNA expression of the novel polypeptide of the present invention was induced very early. MRNA in astrocytes pretreated with cycloheximide, a protein synthesis inhibitor
No change was found in the induction of the expression of, indicating that no new protein synthesis is required for the induction of mRNA expression of the novel polypeptide of the present invention. On the other hand, when pretreated with the NADPH oxidase inhibitor DPI, the expression of the mRNA is clearly suppressed. This means that N
This suggests that active oxygen generated by activation of the ADPH oxidase system directly activates transcription. At this time, in the astrocytes which were subjected to only reoxygenation without hypoxic load, the mR
No induction of NA expression is observed.

Test Example 2 Induction of mRNA of the novel polypeptide of the present invention in various organ tissues Even in organs other than the brain, environmental changes such as hypoxia load and reoxygenation are considered to cause serious problems. Therefore,
The expression of mRNA of the novel polypeptide of the present invention in various organ tissues of rats was examined. Tissues of various organs were excised from rats and total RNA was extracted by the AGPC method. Northern blot analysis was performed in the same manner as in Test Example 1 using the total RNA. The results are shown in FIG. Its expression was confirmed in various organs widely in tissues other than the brain.

Example 2 Preparation of Antibody Specific to the Novel Polypeptide of the Present Invention In order to prepare an antibody having an affinity for the novel polypeptide of the present invention, a sequence corresponding to a part of the amino acid sequence of the polypeptide described below was prepared. Was synthesized using an automatic peptide synthesizer PSSM-8 system (manufactured by Shimadzu Corporation). Cys-Gln-Glu-Asp-Glu-Val-Lys-Arg-Leu-Lys-Ser-Ile- -Met-Gly-Leu-Glu-Gly-Glu-Asp-Glu (SEQ ID NO: 4) Synthetic peptide To BSA and TiterMa
x (manufactured by Vaxel), and the mixture 1 mg / 1 ml
Was immunized by subcutaneous injection into a rabbit (New Zealand white species, 1.5 kg). The peptide B
0.5 mg / 0.5 ml of SA-TiterMax mixture
Was subcutaneously injected and boosted to obtain an antiserum. The antibody titer was confirmed by the reactivity with the peptide antigen.
In order to confirm the affinity between the obtained antiserum and the polypeptide of the present invention, Western blot analysis was performed using the antibody.

First, astrocytes cultured to a confluent state in the same manner as in Example 1 were exposed to hypoxia for 0, 6, 12 and 24 hours to prepare cells. After another 24 hours of hypoxia, the cells were reoxygenated to prepare cells that had passed for 1, 2, 4 and 8 hours, respectively.
Collect cells under hypoxia or reoxygenation in PBS
(Phosphate buffer, 8 g sodium chloride in 1 L, 0.2 g
g potassium chloride, 1.44 g disodium hydrogen phosphate, 0.24 g monopotassium dihydrogen phosphate)
Washed twice. The cells were lysed and extracted with PBS containing 2% SDS. 10 μg of the obtained cell extract was added to 12.5
% SDS-PAGE. After the electrophoresis, the protein in the gel was applied to Immobilon-P membrane (Millipore) for 1 minute.
Transferred overnight at 3V, 4 ° C. After transfer, the film is
Blocking treatment was carried out at 4 ° C. for 24 hours in a Tris buffer (pH 8.0) containing ween 20 and 0.2% casein, followed by reaction with the antiserum obtained above as a primary antibody. As a secondary antibody, a peroxidase-labeled anti-rabbit IgG antibody (manufactured by Organon Technica) was used at 37 ° C.
For 1 hour. Luciferase (manufactured by DuPont) was used as a coloring substrate. The results are shown in FIG. Rabbit antiserum against the synthetic peptide was obtained on SDS-PAGE.
Only in cell extracts from reoxygenated astrocytes, the molecular weight (about 70 K
A single band of Da) was recognized. However, the expression of the polypeptide had disappeared 8 hours after reoxygenation. This indicates that induction of expression by reoxygenation is transient.

Example 3 Evaluation of a Rat Ischemic Brain Model Using the Novel Polypeptide-Specific Antibody of the Present Invention Using male SD rats (body weight: 250 g), Longa et al.
The method of Nagasawa et al. (Longa et al., Stroke (1989) 20, 84-9
1, Nagasawa et al., Stroke (1989) 20, 1037-1043), and a transient unilateral middle cerebral artery occlusion model was prepared. After 2 hours of arterial occlusion, blood flow was resumed, and one hour after reperfusion, the rats were perfused and fixed with a fixing solution containing 4% paraformaldehyde, and the brain was removed. The excised brain was frozen with powdered dry ice, and the thickness was 16 μm using a cryostat (manufactured by Leica).
m frozen thin sections were prepared. The thin section was analyzed using the specific antibody. That is, the section was placed in 0.02 MPBS.
After washing, the specific antibody of the present invention was placed in a solution diluted 20-fold with PBS, and reacted at 4 ° C. for 24 hours (first reaction). After the first reaction, the section was washed with PBS for 30 minutes, exchanging the solution, and washed three times in total. Then, the section was mixed with a biotin-labeled goat anti-rabbit IgG antibody (manufactured by Vector) diluted 500-fold with PBS for 2 hours at room temperature. (Second reaction). After that, similarly washed with PBS, P containing 0.2% hydrogen peroxide
After blocking endogenous peroxidase with BS, an ABC kit (Vectastein Eli) manufactured by Vector is used.
te) to react with a peroxidase-labeled avidin-biotin complex solution (ABC reaction). Then, the section was washed twice with PBS and 0.05 M Tris-HCl buffer (pH 7.6) each, and then dia was added.
The polypeptide was visualized using a minobenzidine (DAB) reaction. The section was affixed on a slide glass with a gelatin solution, dried, dehydrated with ethanol, embedded in Enteran (manufactured by Merck), and observed with an optical microscope.

Using a section adjacent to the brain section subjected to the immunohistochemistry using the above-described specific antibody of the present invention, Microtubul was used.
The staining properties of ar associated protein 2 (MAP-2) were observed. This is because MAP-2 staining disappears very early after ischemia loading in nerve cells after cerebral ischemia loading, which is a good indicator of ischemic neuronal damage (Kitagawa et al., Neuron).
oscience (1989) 31, 401-411). MAP-2 immunohistochemistry was based on that of the specific antibody of the present invention. That is, mouse monoclonal anti-MAP-2 antibody (manufactured by Amersham) was diluted 500-fold in the first reaction, and reacted with biotin-labeled goat anti-mouse IgG antibody (500-fold diluted).
A BC reaction was performed. Adjacent sections stained with this MAP-2 antibody were also subjected to microscopic observation after the DAB reaction. FIG. 4 shows the results. As for the staining property of the polypeptide of the present invention, strong expression was observed in astrocytes of the cerebral cortex layers II-III in the middle cerebral artery region very early, one hour after transient middle cerebral artery occlusion / reperfusion. Arrows in the figure indicate cells expressing the novel polypeptide of the present invention (FIG. 4A).
At this time, MAP-2 of the neural dendrites of the adjacent section in the same region
It is clear that the neurons were subjected to an ischemic load, as indicated by a decrease in staining (FIG. 4B). The expression of the novel polypeptide of the present invention was remarkable in the region subjected to ischemic load, suggesting that it is possible to evaluate cerebral ischemic pathology using the specific antibody of the present invention. .

Test Example 3 Intracellular distribution of the novel polypeptide of the present invention Since the novel polypeptide has homology to vesicle transport proteins such as yeast, it is considered that the novel polypeptide is one of new vesicle transport proteins. In order to examine where in the cells the polypeptide functions, the intracellular distribution in rat primary cultured astrocytes was examined. A hypoxia challenge (24 hours) was used as a cell sample, followed by primary cultured astrocytes of rats 4 hours after reoxygenation.

1) Examination by Western Blot Analysis Each cell was crushed by the Nitrogen Cavity method in PBS containing 1% NP-40 (Nonidet P-40), and the cells were treated with 38, 30 and 20% sucrose solutions. ℃,
Centrifugation at 10,000 g for 3 hours was performed to fractionate each intracellular organ (Kuwabara et al., J. Biol. Chem. (1996) 271, 502).
5-5032). Western blot analysis was performed using the specific antibody of the present invention in the same manner as in Example 2 using the obtained cytoplasmic fraction, plasma membrane fraction, Golgi body fraction and endoplasmic reticulum fraction. The results are shown in FIG. 5A. It can be seen that the novel polypeptide is present in the Golgi fraction in astrocytes 4 hours after reoxygenation.

2) Examination by immunofluorescence staining The cells were fixed with Zamboni solution at room temperature for 2 hours. 0.
After washing with 2MPBS, the immune buffer (5% BSA,
1% NGS, 0.3% Triton X-100, 0.1
(MPBS) at room temperature for 2 hours. Subsequently, the reaction was carried out overnight at 4 ° C. in an immune buffer containing the specific antibody of the present invention. The antiserum prepared in Example 2 was used for the antibody.
On ^™ kit1 (manufactured by Multiple Peptide System)
Was used. After washing with 0.02 M PBS, FITC-labeled anti-rabbit IgG antibody was used as a secondary antibody,
The reaction was performed at 4 ° C. for 2 hours. In addition, in order to examine the positional relationship between the polypeptide and the Golgi apparatus, the distribution of Wet Germ Agglutinin (WGA), which is a Golgi apparatus marker, was similarly examined. The cells pre-fixed as described above were reacted overnight at 4 ° C. with FITC-labeled WGA (Molecular Probes). Fluorescently labeled cells were observed with a fluorescence microscope. As a control, cells to which the synthetic peptide used as the antigen in Example 2 at the time of the reaction with the specific antibody of the present invention was simultaneously added were used. The results are shown in FIG. 5B. The distribution of the polypeptide showed a pattern very similar to the stained image by WGA, and it was confirmed that the novel polypeptide of the present invention was accumulated in the Golgi apparatus.

Test Example 4 Interleukin 6 (IL-
6) Relevance to induction of activity IL-6 in the medium after reoxygenation in astrocytes
It is already known that activity is induced (Maeda et al.,
J. Exp. Med. (1994) 180 2297-2308). Primary culture of astrocytes cultured on 6-well plate
After culturing for 2 hours, reoxygenation and IL-6 activity in the culture supernatant after 0, 2, 4, 8, and 16 hours were measured by the method of Maeda et al.
a et al., J. Exp. Med. (1994) 180 2297-2308).
It was measured in an assay system using H-60 cells. further,
To examine the role of the novel polypeptide of the present invention in inducing IL-6 activity, an antisense oligonucleotide for the polypeptide shown below (the expression of the polypeptide of the present invention is suppressed by the addition of the antisense oligonucleotide) It was confirmed that 10 μM was added to the medium 30 minutes before reoxygenation, and then reoxygenated to 0,
After 2, 4, 8, and 16 hours, the IL-6 activity in the culture supernatant was measured. FIG. 6 shows the results. 5′-CTTCCCACCATTCACTGCC-3 ′ (SEQ ID NO: 5 in Sequence Listing) As shown in FIG. 6, IL-6 activity induction was inhibited by about 70% in astrocytes to which an antisense oligonucleotide was added. As a control, no inhibition of IL-6 activity induction was observed in cells to which the sense oligonucleotide of the antisense oligonucleotide was added. In addition, the addition of antisense oligonucleotides
When the intracellular distribution of IL-6 in astrocytes in which the induction of L-6 activity was inhibited was examined by Western blot analysis, IL-6 was present in the cytoplasm in cells in which IL-6 activity was induced. On the other hand, in cells in which the induction of activity was inhibited by the addition of the antisense oligonucleotide, IL-6 remained accumulated in the Golgi apparatus. From these results, the novel polypeptide of the present invention is IL
It is thought to be involved in the ER-Golgi transport of -6.

[0050]

The novel polypeptide of the present invention is induced to be expressed by hypoxic load / reoxygenation. Therefore, by examining the expression status of the polypeptide of the present invention, it is possible to diagnose, evaluate and elucidate the pathology of ischemia / reperfusion injury occurring in vivo and / or in vitro. More specifically, the expression of the novel polypeptide of the present invention is analyzed at the mRNA level and at the protein level by using a DNA encoding the novel polypeptide and an antibody having an affinity for the novel polypeptide. Can be. Furthermore, these enable screening of new preventive and therapeutic agents for ischemia / reperfusion injury.

[0051]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 637 Sequence type: amino acid Number of chains: 1 chain Sequence type: protein Sequence: Met Val Gly Ser Lys Met Ala Ala Ser Ile Arg Glu Arg Gln Thr Val 1 5 10 15 Ala Leu Lys Arg Met Leu Asn Phe Asn Val Pro His Val Lys Asn Ser 20 25 30 Pro Gly Glu Pro Val Trp Lys Val Leu Ile Tyr Asp Arg Phe Gly Gln 35 40 45 Asp Ile Ile Ser Pro Leu Leu Ser Val Lys Glu Leu Arg Asp Met Gly 50 55 60 Ile Thr Leu His Leu Leu Leu His Ser Asp Arg Asp Pro Ile Arg Asp 65 70 75 80 Val Pro Ala Val Tyr Phe Val Met Pro Thr Glu Glu Asn Ile Asp Arg 85 90 95 Leu Cys Gln Asp Leu Arg Asn Gln Leu Tyr Glu Ser Tyr Tyr Leu Asn 100 105 110 Phe Ile Ser Ala Ile Ser Arg Ser Lys Leu Glu Asp Ile Ala Asn Ala 115 120 125 Ala Leu Ala Ala Asn Ala Val Thr Gln Val Ala Lys Val Phe Asp Gln 130 135 140 Tyr Leu Asn Phe Ile Thr Leu Glu Glu Asp Met Phe Val Leu Cys Asn 145 150 155 160 Gln Asn Lys Glu Leu Val Ser Tyr Arg Ala Ile Asn Arg Pro Asp Ile 165 170 175 Thr Asp Thr Glu Met Glu Thr Val M et Asp Thr Ile Val Asp Ser Leu 180 185 190 Phe Cys Phe Phe Val Thr Leu Gly Ala Val Pro Ile Ile Arg Cys Ser 195 200 205 Arg Gly Thr Ala Ala Glu Met Val Ala Val Lys Leu Asp Lys Lys Leu 210 215 220 Arg Glu Asn Leu Arg Asp Ala Arg Asn Ser Leu Phe Thr Gly Asp Pro 225 230 235 240 Leu Gly Thr Gly Gln Phe Ser Phe Gln Arg Pro Leu Leu Val Leu Val 245 250 255 Asp Arg Asn Ile Asp Leu Ala Thr Pro Leu His His Thr Trp Thr Tyr 260 265 270 Gln Ala Leu Val His Asp Val Leu Asp Phe His Leu Asn Arg Val Asn 275 280 285 Leu Glu Glu Ser Thr Gly Val Glu Asn Ser Pro Thr Gly Ala Arg Pro 290 295 300 Lys Arg Lys Asn Lys Lys Ser Tyr Asp Leu Thr Pro Val Asp Lys Phe 305 310 315 320 Trp Gln Lys His Lys Gly Ser Pro Phe Pro Glu Val Ala Glu Ser Val 325 330 335 Gln Gln Glu Leu Glu Ser Tyr Arg Ala Gln Glu Asp Glu Val Lys Arg 340 345 350 Leu Lys Ser Ile Met Gly Leu Glu Gly Glu Asp Glu Gly Ala Ile Ser 355 360 365 Met Leu Ser Asp Asn Thr Ala Lys Leu Thr Ser Ala Val Ser Ser Leu 370 375 380 Pro Glu Leu Leu Glu Lys Lys Arg LeuIle Asp Leu His Thr Asn Val 385 390 395 400 Ala Thr Ala Val Leu Glu His Ile Lys Ala Arg Lys Leu Asp Val Tyr 405 410 415 Phe Glu Tyr Glu Glu Lys Ile Met Ser Lys Thr Thr Leu Asp Lys Ser 420 425 430 Leu Leu Asp Val Ile Ser Asp Pro Asp Ala Gly Thr Pro Glu Asp Lys 435 440 445 Met Arg Leu Phe Leu Ile Tyr Tyr Ile Ser Ala Gln Gln Ala Pro Ser 450 455 460 Glu Val Asp Leu Glu Gln Tyr Lys Lys Ala Leu Thr Asp Ala Gly Cys 465 470 475 480 480 Asn Leu Ser Pro Leu Gln Tyr Ile Lys Gln Trp Lys Ala Phe Ala Lys 485 490 495 Met Ala Ser Thr Pro Ala Ser Tyr Gly Asn Thr Thr Thr Lys Pro Met 500 505 510 Gly Leu Leu Ser Arg Val Met Asn Thr Gly Ser Gln Phe Val Met Glu 515 520 525 Gly Val Lys Asn Leu Val Leu Lys Gln Gln Asn Leu Pro Val Thr Arg 530 535 540 Ile Leu Asp Asn Leu Met Glu Met Lys Ser Asnn Pro Glu Thr Asp Asp 545 550 555 560 Tyr Arg Tyr Phe Asp Pro Lys Met Leu Arg Ser Asn Asp Ser Ser Val 565 570 575 Pro Arg Asn Lys Ser Pro Phe Gln Glu Ala Ile Val Phe Val Val Gly 580 585 585 590 Gly Gly Asn Tyr Ile Glu Tyr Gln AsnLeu Val Asp Tyr Ile Lys Gly 595 600 605 Lys Gln Gly Lys His Ile Leu Tyr Gly Cys Ser Glu Ile Phe Asn Ala 610 615 620 Thr Gln Phe Ile Lys Gln Leu Ser Gln Leu Gly Gln Lys 625 630 635 637

SEQ ID NO: 2 Sequence length: 2095 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence AGCCGGGCAG TGA ATG GTG GGA AGC AAG ATG GCG GCC AGC ATT CGG GAA AGG 52 Met Val Gly Ser Lys Met Ala Ala Ser Ile Arg Glu Arg 1 5 10 CAA ACA GTG GCT CTG AAG CGG ATG TTG AAT TTC AAT GTG CCT CAT GTT 100 Gln Thr Val Ala Leu Lys Arg Met Leu Asn Phe Asn Val Pro His Val 15 20 25 AAA AAC AGT CCT GGA GAA CCC GTA TGG AAG GTA CTC ATC TAT GAC AGA 148 Lys Asn Ser Pro Gly Glu Pro Val Trp Lys Val Leu Ile Tyr Asp Arg 30 35 40 45 TTT GGC CAA GAT ATC ATC TCT CCT CTG CTG TCT GTG AAG GAG CTG AGA 196 Phe Gly Gln Asp Ile Ile Ser Pro Leu Leu Ser Val Lys Glu Leu Arg 50 55 60 GAC ATG GGC ATC ACC CTG CAT CTC CTT TTG CAC TCA GAC CGA GAT CCA 244 Asp Met Gly Ile Thr Leu His Leu Leu Leu His Ser Asp Arg Asp Pro 65 70 75 ATT CGA GAT GTT CCT GCG GTG TAC TTT GTG ATG CCA ACC GAA GAA AAT 292 Ile Arg Asp Val Pro Ala Val Tyr Phe Val Met Pro Thr Glu Glu Asn 80 85 90 ATT GAC AGA CTG TGC CAG GAT CTT CGA AAT CAG CTC TAT GAA TCC TAT 340 Ile Asp Arg Leu Cys Gln Asp Leu Arg Asn Gln Leu Tyr Glu Ser Tyr 95 100 105 TAT TTA AAT TTT ATT TCT GCG ATT TCA AGA AGT AAA CTG GAA GAC ATT 388 Tyr Leu Asn Phe Ile Ser Ala Ile Ser Arg Ser Lys Leu Glu Asp Ile 110 115 120 125 GCA AAT GCA GCA TTG GCC GCT AAT GCA GTC ACA CAG GTT GCC AAG GTT 436 Ala Asn Ala Ala Leu Ala Ala Asn Ala Val Thr Gln Val Ala Lys Val 130 135 140 TTT GAC CAG TAT CTC AAT TTT ATT ACT TTG GAA GAG GAC ATG TTT GTA 484 Phe Asp Gln Tyr Leu Asn Phe Ile Thr Leu Glu Glu Asp Met Phe Val 145 150 155 TTA TGT AAT CAA AAT AAG GAA CTT GTT TCA TAT CGG GCC ATT AAT AGG 532 Leu Cys Asn Gln Asn Lys Glu Leu Val Ser Tyr Arg Ala Ile Asn Arg 160 165 170 CCA GAT ATC ACA GAC ACA GAG ATG GAG ACT GTT ATG GAC ACT ATT GTT 580 Pro Asp Ile Thr Asp Thr Glu Met Glu Thr Val Met Asp Thr Ile Val 175 180 185 GAC AGC CTC TTC TGC TTT TTT GTT ACA TTA GGT GCT GTT CCC ATC ATC 628 Asp Ser Leu Phe Cys Phe Phe Val Thr Leu Gly Ala Val Pro Il e Ile 190 195 200 205 CGA TGC TCA AGA GGA ACG GCA GCA GAA ATG GTG GCA GTG AAA CTA GAT 676 Arg Cys Ser Arg Gly Thr Ala Ala Glu Met Val Ala Val Lys Leu Asp 210 215 220 220 AAA AAA CTG CGG GAG AAT CTA AGA GAT GCA AGA AAC AGC CTT TTT ACT 724 Lys Lys Leu Arg Glu Asn Leu Arg Asp Ala Arg Asn Ser Leu Phe Thr 225 230 235 GGT GAT CCA CTT GGG ACT GGC CAG TTC AGC TTC CAA AGG CCC TTA TTA 772 Gly Asp Pro Leu Gly Thr Gly Gln Phe Ser Phe Gln Arg Pro Leu Leu 240 245 250 GTC CTT GTG GAC AGA AAC ATT GAC TTG GCA ACG CCT CTG CAC CAT ACG 820 Val Leu Val Asp Arg Asn Ile Asp Leu Ala Thr Pro Leu His His Thr 255 260 265 TGG ACA TAC CAA GCG CTG GTA CAC GAT GTC CTG GAT TTC CAC TTA AAC 868 Trp Thr Tyr Gln Ala Leu Val His Asp Val Leu Asp Phe His Leu Asn 270 275 275 280 285 AGA GTA AAT TTG GAA GAA TCT ACA GGA GTG GAA AAT TCT CCA ACT GGT 916 Arg Val Asn Leu Glu Glu Ser Thr Gly Val Glu Asn Ser Pro Thr Gly 290 295 300 GCT AGA CCA AAG AGG AAA AAC AAG AAG TCT TAC GAT TTA ACT CCA GTT 964 Ala Arg Pro Lys Arg Lys Asn Lys Lys Ser T yr Asp Leu Thr Pro Val 305 310 315 GAT AAA TTT TGG CAG AAA CAT AAA GGA AGT CCA TTC CCA GAA GTC GCA 1012 Asp Lys Phe Trp Gln Lys His Lys Gly Ser Pro Phe Pro Glu Val Ala 320 325 330 GAA TCA GTC CAA CAA GAA CTA GAA TCT TAC AGA GCA CAA GAA GAT GAG 1060 Glu Ser Val Gln Gln Glu Leu Glu Ser Tyr Arg Ala Gln Glu Asp Glu 335 340 345 GTC AAA CGA CTG AAG AGC ATT ATG GGC CTA GAA GGA GAG GAC GAA GGA 1108 Val Lys Arg Leu Lys Ser Ile Met Gly Leu Glu Gly Glu Asp Glu Gly 350 355 360 365 GCC ATC AGC ATG CTT TCT GAT AAC ACT GCT AAG CTC ACA TCA GCT GTC 1156 Ala Ile Ser Met Leu Ser Asp Asn Thr Ala Lys Leu Thr Ser Ala Val 370 375 380 AGT TCT TTG CCA GAA CTC CTT GAA AAA AAA AGA CTT ATC GAT CTC CAT 1204 Ser Ser Leu Pro Glu Leu Leu Glu Lys Lys Arg Leu Ile Asp Leu His 385 390 395 ACA AAT GTC GCC ACT GCT GTT TTA GAA CAC ATA AAG GCA AGA AAA CTG 1252 Thr Asn Val Ala Thr Ala Val Leu Glu His Ile Lys Ala Arg Lys Leu 400 405 410 GAT GTA TAT TTT GAA TAT GAA GAA AAA ATA ATG AGC AAG ACT ACT CTG 1300 Asp Val Tyr Phe Glu Ty r Glu Glu Lys Ile Met Ser Lys Thr Thr Leu 415 420 425 GAT AAG TCC CTT CTC GAC GTC ATA TCT GAC CCT GAC GCA GGG ACT CCG 1348 Asp Lys Ser Leu Leu Asp Val Ile Ser Asp Pro Asp Ala Gly Thr Pro 430 435 440 445 GAA GAC AAA ATG AGG CTG TTT CTT ATC TAC TAC ATA AGC GCT CAG CAG 1396 Glu Asp Lys Met Arg Leu Phe Leu Ile Tyr Tyr Ile Ser Ala Gln Gln 450 455 460 GCA CCA TCT GAG GTT GAT TTG GAG CAG TAT AAA AAG GCT TTA ACA GAT 1444 Ala Pro Ser Glu Val Asp Leu Glu Gln Tyr Lys Lys Ala Leu Thr Asp 465 470 475 GCA GGA TGC AAC CTT AGC CCT TTA CAG TAT ATC AAA CAG TGG AAG GCT 1492 Ala Gly Cys Asn Leu Ser Pro Leu Gln Tyr Ile Lys Gln Trp Lys Ala 480 485 490 TTT GCC AAG ATG GCC TCA ACT CCT GCC AGC TAC GGA AAC ACT ACC ACT 1540 Phe Ala Lys Met Ala Ser Thr Pro Ala Ser Tyr Gly Asn Thr Thr Thr 495 500 505 AAA CCA ATG GGT CTC TTG TCC CGA GTC ATG AAT ACA GGA TCC CAG TTT 1588 Lys Pro Met Gly Leu Leu Ser Arg Val Met Asn Thr Gly Ser Gln Phe 510 515 520 525 GTG ATG GAA GGC GTC AAG AAC CTG GTA TTG AAG CAG CAG AAT CTA CCT 1636 Val Met Glu Gly Val Lys Asn Leu Val Leu Lys Gln Gln Asn Leu Pro 530 535 540 GTT ACT CGG ATT TTA GAC AAT CTC ATG GAG ATG AAG TCA AAC CCC GAG 1684 Val Thr Arg Ile Leu Asp Asn Leu Met Glu Met Lys Ser Asn Pro Glu 545 550 555 ACT GAT GAT TAC AGA TAT TTT GAT CCC AAA ATG CTG CGG AGC AAT GAC 1732 Thr Asp Asp Tyr Arg Tyr Phe Asp Pro Lys Met Leu Arg Ser Asn Asp 560 565 570 570 AGC TCA GTT CCT AGG AAC AAA AGT CCA TTC CAA GAG GCC ATT GTC TTT 1780 Ser Ser Val Pro Arg Asn Lys Ser Pro Phe Gln Glu Ala Ile Val Phe 575 580 585 GTG GTA GGA GGA GGC AAC TAT ATT GAG TAT CAG AAT CTT GTT GAC TAC 1828 Val Val Gly Gly Gly Asn Tyr Ile Glu Tyr Gln Asn Leu Val Asp Tyr 590 595 600 605 ATA AAG GGA AAG CAA GGC AAG CAT ATT TTG TAT GGC TGC AGT GAG ATT 1876 Ile Lys Gly Lys Gln Gly Lys His Ile Leu Tyr Gly Cys Ser Glu Ile 610 615 620 TTT AAT GCT ACA CAG TTC ATA AAA CAG CTG TCA CAG CTT GGA CAA AAG 1924 Phe Asn Ala Thr Gln Phe Ile Lys Gln Leu Ser Gln Leu Gly Gln Lys 625 630 635 637 TAACACAGAA GAGTCATAAT GGGTGATCAG TGTGGAGAGA T GTAAAAAGC CAGACGTGTC 1984 CTTCTCCATA GCAGTGCCCT AACAGTGCAA CCTGCGGAAT CAGTCATTTT TAAAGAAATT 2044 CTATACTTCA TATACTGTAC AATGATTAAA ATAATAAACC ATTTCAGAAG T 2095

SEQ ID NO: 3 Sequence length: 12 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Chemically synthesized DNA Sequence: TGGTACGGTA TA 12

SEQ ID NO: 4 Sequence length: 20 Sequence type: number of amino acid chains: single chain Sequence type: peptide Sequence: Cys Gln Glu Asp Glu Val Lys Arg Leu Lys Ser Ile Met Gly Leu Glu 15 10 15 Gly Glu Asp Glu 20

SEQ ID NO: 5 Sequence length: 19 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Chemically synthesized DNA Sequence: CTTCCCACCA TTCACTGCC 19

[Brief description of the drawings]

FIG. 1. mR of the novel polypeptide of the present invention by hypoxic load and reoxygenation in primary cultured astrocytes of rats
4 is a Northern blot analysis image (electrophoresis photograph) instead of a drawing showing induction of NA expression.

FIG. 2 is a Northern blot analysis image (electrophoresis photograph) instead of a drawing, showing the expression status of mRNA of the novel polypeptide of the present invention in various organ tissues of rats.

FIG. 3 is a western blot analysis image (electrophoresis photograph) showing induction of expression of the novel polypeptide of the present invention by hypoxia load / reoxygenation in primary cultured astrocytes of rats.
It is.

FIG. 4A is an immunohistologically stained image (photograph of the morphology of an organism) showing the induction of expression of the novel polypeptide of the present invention in a rat model of transient unilateral middle cerebral artery occlusion in a rat.
Arrows indicate cells expressing the novel polypeptide of the present invention. B: Immunohistochemical staining image (morphological photograph of an organism) showing a distribution of MAP-2 in a rat model of transient unilateral middle cerebral artery occlusion, which is a drawing.

FIG. 5A: Western blot analysis image (electrophoresis photograph) instead of a drawing showing the intracellular distribution of the novel polypeptide of the present invention in primary cultured astrocytes of hypoxia-loaded / reoxygenated rat primary culture. B: Fluorescence microscopy image (photograph of the morphology of cells) showing the intracellular distribution of the novel polypeptide of the present invention in primary cultured astrocytes of hypoxia-loaded / reoxygenated rat.

FIG. 6 is a graph showing the effect of the antisense oligonucleotide of the novel polypeptide of the present invention on the induction of IL-6 activity by reoxygenation in primary cultured astrocytes of rats.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication location C12N 15/09 ZNA C12P 21/08 C12P 21/02 C12N 5/00 B 21/08 9282-4B 15 / 00 ZNAA (72) Inventor Satoshi Ogawa 77, Momoyama-Nagaoka-Ecchu-Minamicho, Fushimi-ku, Kyoto-shi, Kyoto (72) Inventor Akio Wanaka 7-10-2 Horaicho, Fukushima-shi, Fukushima 2F

Claims (17)

[Claims] 1. A polypeptide having an amino acid sequence substantially as shown in SEQ ID NO: 1 in the Sequence Listing, and whose expression is induced upon reoxygenation after hypoxic load. 2. The polypeptide according to claim 1, wherein the reoxygenation after hypoxic load is in the form of reperfusion after ischemia. 3. The polypeptide according to claim 1, which is derived from a mammal. 4. The polypeptide according to claim 1, which is derived from a rat. A DNA encoding the novel polypeptide according to any one of claims 1 to 4. 6. A DNA having substantially the nucleotide sequence of at least nucleotides 14 to 1924 in the nucleotide sequence of SEQ ID NO: 2 in the Sequence Listing. 7. A recombinant vector containing the DNA according to claim 5 or 6. 8. A host cell transformed with the recombinant vector according to claim 7. 9. The transformed cell according to claim 8, wherein the host cell is an animal cell. 10. The method according to claim 1, wherein the host cell according to claim 8 or 9 is cultured, and the polypeptide according to claim 1 is collected from the obtained culture.
A method for producing a polypeptide according to any one of claims 1 to 4. 11. An antibody exhibiting affinity for the polypeptide according to claim 1. 12. An antibody exhibiting affinity for a peptide having substantially all or a part of the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing. 13. An antibody exhibiting affinity for a peptide having at least the amino acid sequence represented by amino acid numbers 346 to 364 in the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. 14. A method for evaluating an ischemia / reperfusion injury model, comprising using the antibody according to claim 11. 15. A kit for evaluating and diagnosing ischemia / reperfusion injury, comprising the antibody according to claim 11. 16. A method for screening a drug for preventing and treating ischemia / reperfusion injury, which comprises using the antibody according to claim 11. 17. A kit for screening for a prophylactic and therapeutic agent for ischemia / reperfusion injury, comprising the antibody according to claim 11.